Targeted photodynamic therapy for breast cancer: the potential of glyconanoparticles

Photodynamic therapy (PDT) uses a non-toxic light sensitive molecule, a photosensitiser, that releases cytotoxic reactive oxygen species upon activation with light of a specific wavelength. Here, glycan-modified 16 nm gold nanoparticles (glycoAuNPs) were explored for their use in targeted PDT, where the photosensitiser was localised to the target cell through selective glycan–lectin interactions. Polyacrylamide (PAA)-glycans were chosen to assess glycan binding to the cell lines. These PAA-glycans indicated the selective uptake of a galactose-derivative PAA by two breast cancer cell lines, SK-BR-3 and MDA-MD-231. Subsequently, AuNPs were modified with a galactose-derivative ligand and an amine derivate of the photosensitiser chlorin e6 was incorporated to the nanoparticle surface via amide bond formation using EDC/NHS coupling chemistry. The dual modified nanoparticles were investigated for the targeted cell killing of breast cancer cells, demonstrating the versatility of using glycoAuNPs for selective binding to different cancer cells and their potential use for targeted PDT.


General
All reagents were of analytical grade and purchased from Sigma Aldrich, Merck, Fischer Scientific or Thermo Fischer Scientific, unless stated otherwise.PAA-glycans (with molecular weight ca.2,800 g/mol) were purchased from GlycoTech.The SK-BR-3 cell line was kindly provided by Professor Dylan Edwards (University of East Anglia, UK), which was obtained from LGC Standards.MDA-MB-231 and MCF-10A were purchased from ATCC (LGC Standards).

Mass spectrometry
Two mass spectrometry methods were used.Firstly, ESI-MS was used during ligand synthesis (Advion Expression Compact Mass Spectrometer).Through direct injection, 10 µL of ca.0.1 mg•mL -1 was injected.The masses were recorded in positive mode and analysed using Advion Mass Express software.Secondly, MALDI-TOF MS analysis was used (Bruker Daltonics autoflex speed ToF/ToF mass spectrometer).Concentrated samples (ca.150 nM AuNPs) were prepared by performing a 1: 1 dilution of sample in DHB matrix, and then 2 µL of the mixture was spotted onto a MTP AnchorChip 384 target plate.The spot was left to dry at room temperature before analysis.The MALDI-TOF MS equipment used a nitrogen laser, and analysis was performed in a linear 50 shot mode, with 32x gain and 70% laser intensity.

Nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance (NRM) was performed in a Bruker Avange III HD 400 MHz.The instrument used a broadband BBFO probe at 400 MHz (1H) and 100 MHz (13C) at 298 °C.For the analysis, the chemical shifts (δ) are in parts per million (ppm).Compounds were assigned using proton, carbon, HSQC edited two-dimensional correlation spectroscopy and COSY.Assignment was performed using Mestrenova software (Mestrelab Research, S.L.).

UV-Vis spectroscopy
UV-Vis measurements were obtained using a Varian Cary® 50 UV-Vis spectrophotometer or a BMG Labtech CLARIOstar® high-performance microplate reader.For the UV-Vis spectrophotometer measurements, samples (1 mL) were added to a quartz cuvette with a 1 cm path length and measured using Cary WinUV software.For the plate reader measurements, samples (50 µL) were loaded into 384-well microtitre plates (4titude), and measurements recorded using Omega series and MARS Data Analysis software (BMG Labtech).

Fluorimetry
Fluorescence measurements were obtained using a Hitachi F-4500 fluorimeter or a BMG Labtech CLARIOstar® high-performance microplate reader.For the fluorimeter measurements, samples (1 mL) were added to a Quartz cuvette with a 1 cm path length, and measurements were recorded using FluorEssence TM (Horiba) software.For the plate reader measurements, samples (120 µL) were loaded into Nunc Nunclon TM Surface 96-well microtitre plates, and measurement were recorded using Omega series and MARS Data Analysis software (BMG Labtech).

Confocal laser scanning microscopy and image analysis
A Carl Zeiss LSM 510 META confocal laser scanning microscope with a plan-apochromat 63x/1.4Oil DIC objective was used to obtain the images, and the data was processed using ImageJ/Fĳi software.For quantification of fluorescence, each image was despeckled, followed by setting the minimum threshold to 23, with the 'Triangle' threshold method.The threshold was determined using the condition which provided the highest grey value, i.e., the strongest interaction.The threshold was determined at the point where all background signal was removed and only fluorescence due to the dye interaction with the cell was measured.The threshold was then applied throughout all images for analysis.Fluorescence within the cells was represented and quantified by the integrated intensity, which was determined as follows.The integrated intensity of one image is derived from the following equation (Eq.1): (Eq. 1)

𝑛
Where the mean grey value is the average intensity of all pixels measured.The area is the total area of the selection within the image where the grey values were recorded, and n is the number of cells within that image.
The mean integrated intensity is consequently calculated using Eq.2: (Eq. 2) Where is the sum of all integrated intensity for all images within one condition, and n is the ∑  number of images analysed using that condition.

Buffers and media
The composition of the buffers and media supplements used in this research is summarised in Table S1.The pH of each buffer was corrected with 1 M NaOH or 1 M HCl.All buffers and media that were used for cell culture were autoclaved at 110 °C for 10 minutes, and sterilised using a 0.22 µm syringe filter (Millex GP).

Human cell lines culture
All cell culture was performed under sterile conditions.The cancer cell lines, MDA-MB-231 and SK-BR-3, were cultured in DMEM(+), and the MCF-10A cell line was cultured in MEGM.All reagents were heated to 37 °C before addition to the cells.

Starting new cell cultures
Vials of the three cell lines were removed from storage in liquid nitrogen.The cells were thawed at room temperature.To the thawed cells, 1 mL of media was added, and using a pipette, the cells were gently mixed to ensure dispersion into the media.The cells were then transferred to 75 cm 2 Nunc Easy flasks, and a further 10 mL of media was added to make a total volume of 12 mL.All the flasks were then incubated at 37 °C, 5% CO 2 atmosphere.After 24 hours, the media was removed and replenished with 12 mL of fresh media to remove any residual DMSO from the freezing medium.

Subculturing
The cells were subcultured every five days.The cell culture media was discarded and washed with 5 mL of PBS.Next, 5 mL of 0.25% trypsin EDTA was added to the flasks and incubated at 37 °C for 5 mins.The flasks were tapped, to dislodge the cells.To quench the trypsin, 5 mL of DMEM(+) was added to the flasks containing the cancer cell lines.For the MCF-10A cell line, 5 mL of soybean trypsin inhibitor solution (1 mg•mL -1 , in PBS) was added to quench the trypsin.The solutions were added to a 15 mL falcon tube and centrifuged at 800 rcf for 5 mins (MDA-MB-231 and SK-BR-3), or 130 rcf for 7 mins (MCF-10A).The pellets were resuspended in 10 mL of media.The cultures were then diluted 1: 4 in media, transferred into fresh culture flasks, and incubated at 37 °C at 5% CO 2 .

Table S1 :
List of buffers and their composition.